Eye infections caused by Herpes simplex virus affect from 300,000 to 500,000 people each year in the U.S. Infections range in severity from conjunctivitis or mild epithelial keratitis to severe necrotizing keratitis and keratouveitis. These severe infections can cause corneal scarring, vascularization, and corneal clouding leading to blindness or sever reductions in visual ability. Herpes simplex infections are the major cause of blindness due to infectious disease in the U.S. The strain of virus, the host immune response and innate resistance of the host all play a role in determining the severity of the infection. The experiments proposed in this grant application are designed to identify which viral factor or factors (genes) are involved in the development of severe keratitis, to determine which HSV proteins are present in the cornea at the peak of severe eye disease, and to determine which proteins are interacting with the immune system. We have identified two clinical isolates which cause distinct types of eye infections. Isolate 347 causes mild epithelial keratitis and isolate 412 causes severe necrotizing stromal keratitis in BALB/c mice. We will use a variation of marker rescue to transfer cloned DNA from isolate 412 into isolate 347. We will then test the recombinants for their disease pattern. A recombinant virus that has acquired the "virulence" trait from strain 412 will induce severe eye disease allowing us to map its location on the genome. We will identify HSV proteins present in the cornea at the peak of severe eye disease using antisera specific for individual HSV proteins as probes for immunoblots of infected corneal tissues. The HSV glycoproteins will be analyzed first. HSV proteins interacting with the immune system will be identified by using sera from infected BALB/cJ mice to probe immunoblots of viral proteins or to immunoprecipitate proteins from infected cell cultures followed by electrophoresis in SDS-polyacrylamide gels. These approaches are complementary and will provide significant information on the mechanism of development of severe eye disease caused by Herpes simplex virus infections.